Characterization of bee pollen

1. Proceedings of the Third International Conference on Mathematics and Natural Sciences
(ICMNS 2010)
CHARACTERIZATION OF BEE POLLEN FROM
RANCA BUNGUR, BOGOR
Devi Kamilia A., Sukrasno and Irda Fidrianny
School of Pharmacy, Institut Teknologi Bandung
West Java, Indonesia
Abstract. Bee pollen is an agglomerate of pollen grains from various botanical
sources, which are collected by honeybees as their food sources. Bee pollen
contains nutrients, which is the reason why it is used by humans as food
supplement. The objective of this study was to characterize bee pollen that was
obtained from a bee farm in Ranca Bungur, Bogor. The characterization include
major composing pollen, carbohydrate, protein, and lipid contents,
chromatographic pattern, and the main secondary metabolite. Total
carbohydrates content was determined using orcinol-sulphuric acid assay.
Total proteins content was determined using Kjeldahl method. Total lipids
content was determined using Soxhlet extraction method with n-hexane as non
polar solvent, the extract was determined in gravimetric as crude lipid. Bee
pollen was extracted by maceration method with gradual solvent n-hexane,
ethyl acetate, and ethanol. N -hexane extract was fractionated by column classic
chromatography with n-hexane – ethyl acetate (9 : 1) as the eluent. The pure
isolate was characterized using spectrophotometer UV-visible and
spectrophotometer infrared. Macroscopic and microscopic analysis showed the
maize pollen as the major component of bee pollen from Ranca Bungur, Bo gor.
Carbohydrate, protein and lipid contents of the bee pollen were 56.14%,
25.82%, and 5.07% respectively. Total carbohydrate content of bee pollen was
much higher than maize pollen, that were 56.14% and 34.7%. Carbohydrate
components of bee pollen were fructose, glucose , sucrose and other
oligosaccharides, whereas in maize pollen were glucose and fructose.
Phytochemical screening showed positive result for flavonoid and
steroid/triterpenoid. Based on TLC chromatogram with a specific reagent, UV
and IR spectra showed that an isolate obtained from n-hexane extract was
phenolic compound with hydroxyl, carbonyl, aliphatic and aromatic groups.
Keywords: bee pollen, maize pollen, honeybee, carbohydrate, protein, lipid
1 Introduction
Apitherapy began in mainland China and the Middle East, especially Egypt.
Traditional medicine in China has thousands of years before the age of modern
medicine began to rise in Europe. One form of modification of the popular
acupuncture in the world today is by using a needle called honey bee stings bee
acupuncture (bee sting prick). In Indonesia, bee therapy has been conducted since
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2. DEVI KAMILIA A., SUKRASNO & IRDA FIDRIANNY
the 1980s. Apitherapy is a therapy that uses bee products like honey, propolis,
royal jelly, bee venom, and bee pollen. Apis mellifera is the kind of honey bee that
commonly cultivated because of the ability to produce bee products in large
numbers.
Bee pollen is an agglomerate of pollen grains from various botanical sources, which
are collected by the bees and mixed with nectar and secretion from the
hypopharyngeal glands, such as α and β-glycosidase enzymes. Pollen contains
nutrients, such as carbohydrates, proteins, amino acids, lipids, vitamins and
minerals, in addition to carotenoids, flavonoids and phytosterols, which is the
reason why it is used by humans as an alternative food source and/or food
supplement [4].
Bee pollen is used by people as a food supplement and has been reported to have
several pharmacological effects. However, there is insufficient knowledge about the
relationship between chemical constituents with pharmacological effects are
produced. In addition, the chemical content of bee pollen varies and depends on
the condition and biodiversity in the area of cultivated honey bees. Therefore, this
research aims to study the content of nutrients and secondary metabolites
contained in bee pollen, both qualitatively and quantitatively. In addition, from this
study are expected to know the nutritional quality of bee pollen in Indonesia
through analysis of levels of nutrients such as carbohydrates, proteins, and lipids.
2 Materials and methods
Research methods include preparation of materials, inspection characteristics,
determination of nutrient content, and isolation and identification of secondary
metabolites.
The materials used are derived from a bee farm in the area Ranca Bungur, Bo gor
on February 2010. Bee pollen was collected by pollen traps placed at the entrance
of beehives. Maize pollen which allegedly is the main source of bee pollen on the
bee farm is also collected.
Examination includes examining the characteristics of macroscopic, microscopic,
water content, loss on drying, water-soluble extract levels, ethanol-soluble extract
levels, and total ash content. Microscopic examination conducted by the color of
pellets of the results of macroscopic examination.
Determination of nutritional undertaken include: carbohydrates, protein, and lipid.
Determination of total carbohydrate content was done by colorimetric method
using orsinol-sulfuric acid reagent. Determination of total protein content was
done by Kjeldahl method. Determination of total lipid content was done by Soxhlet
equipment using the solvent n-hexane and the weight of extract was determined in
gravimetric as crude lipid.
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3. Characterization Of Bee Pollen From Ranca Bungur, Bogor
Isolation of secondary metabolites was done by extracting with maceration method
using solvents n-hexane, ethyl acetate, and ethanol. The extraction was done on
bee pollen and maize pollen. Monitoring extract performed with thin layer
chromatography (TLC). Monitoring extracts were made to each extract to see the
patterns of chromatograms on bee pollen and maize pollen. N-hexane extract of bee
pollen was fractionated with classical column chromatography using eluent n -
hexane - ethyl acetate (9 : 1). Monitoring of fractions was done by TLC
method. Pure isolates were characterized by using a UV-visible spectrophotometer
and infrared spectrophotometer. The identification was done by using specific
reagents for flavonoid, phenolic, terpenoid, and steroid/triterpenoid.
3 Results and discussion
Bee pollen was collected by honey bees from plants, i.e. pollen from the flowers.
Therefore, bee pollen can be categorized as plant exudates. Bee pollen was
collected in the honey bee farms in the area Ranca Bungur, Bogor, Indonesia in
February 2010. The collection of bee pollen was done by installing a pollen trap at
the entrance of hives that when worker bees were collecting bee pollen about to
enter the hive, bee pollen was carried in baskets corbiculae pollen found on his
hind legs will fall. Bee pollen stored in containers that are stored in the bottom of
the appliance trap pollen. Bee pollen was collected in the form of pellets that vary
in color. Bee pollen dried with a cold and are not exposed to direct sunlight, then
stored in the freezer, to keep the bee pollen remain durable.
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4. DEVI KAMILIA A., SUKRASNO & IRDA FIDRIANNY
Figure 1 Pollen trap placed at the entrance of beehive and bee pollen collected
in the form of pellet.
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5. Characterization Of Bee Pollen From Ranca Bungur, Bogor
By macroscopic, bee pollen form was pellets that vary in colors: dark yellow, white,
brown, orange, and yellow. Then, the microscopic examination of bee pollen pellets
was done based on these colors.
(i) (ii) (iii)
(iv) (v)
Figure 2 Result of macroscopic examination of bee pollen by color of pellets: (i) dark
yellow, (ii) white, (iii) orange, (iv) yellow, and (v) brown.
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6. DEVI KAMILIA A., SUKRASNO & IRDA FIDRIANNY
(i) (ii) (iii)
(iv) (v)
Figure 3 Result of microscopic examination of bee pollen by color of pellets: (i) dark
yellow, (ii) white, (iii) orange, (iv) yellow, and (v) brown.
From the image can be seen that honey bees collect pollen from various plants.
However, the majority of bee pollen pellets was dark yellow that after identified
microscopically containing maize pollen.
Figure 4 Result of microscopic examination of the maize pollen.
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7. Characterization Of Bee Pollen From Ranca Bungur, Bogor
Table 1 Quality Characteristics of Bee Pollen
Characteristics Result (%)
Water content 10.00
Water-soluble extract levels 36.55
Ethanol-soluble extract levels 15.95
Total ash content 1.88
Loss on drying 14.47
Table 2 Nutritional Contents of Bee Pollen
Nutritional content Bee pollen (%) Maize pollen (%)
Carbohydrate 56.14 ± 0.07 34.70 ± 0.09
Crude protein 25.82 ± 0.34 -
Lipid 5.07 ± 0.12 -
1 2 3 4 5 6 7 8 9
Figure 5 Paper chromatogram of water extract of bee pollen and water extract of maize pollen,
mobile phase n-butanol - pyridine - water (10 : 3 : 3), diphenylamine - aniline -
phosphoric acid spray reagent, (1) arabinose, (2) fructose, ( 3) galactose, (4) glucose, (5)
maltose, (6) rhamnose, (7) sucrose, (8) water extract of maize pollen, (9) water extract
of bee pollen
From the results of determination of total carbohydrate content showed that bee
pollen contains more carbohydrate than the pollen of maize. This showed the
addition of carbohydrates in bee pollen. Carbohydrates were likely derived from the
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8. DEVI KAMILIA A., SUKRASNO & IRDA FIDRIANNY
nectar which is also carried by honey bees. Next step was the identification of the
sugar compounds found in bee pollen and maize pollen by paper chromatography
using the comparison compounds are sugar derivatives of arabinose, fructose,
galactose, glucose, rhamnose, maltose, and sucrose. Mobile phase used was n-
butanol - pyridine - water (10 : 3 : 3) and reagent used were diphenylamine -
aniline - phosphoric acid. Extracts tested were water extract of bee pollen and
water extract of maize pollen.
From the results of paper chromatography can be seen that the extract of bee
pollen contains more sugar. Also, it can be seen that the carbohydrate component
in bee pollen include fructose, glucose, sucrose and other oligosaccharides,
whereas in maize pollen contained only glucose and fructose, with fructose as the
main component.
Isolation of secondary metabolites involves several stages, at the beginning of
phytochemical screening experiments performed to determine the class of
compounds contained in bee pollen. The phytochemical screening showed that bee
pollen contains flavonoids and steroid/triterpenoid.
Table 3 Phytochemical Screening of Bee Pollen
Golongan senyawa Hasil
Flavonoid +
Tannin -
Alkaloid -
Quinone -
Saponin -
Steroid/Triterpenoid +
Bee pollen and maize pollen was extracted by maceration method gradually in 3
solvent with increasing polarity. After extraction, monitoring of the extract
performed by thin layer chromatography (TLC) with the stationary phase of silica
gel GF254. Mobile phase used for each extract n-hexane, ethyl acetate, and ethanol
in succession was toluene - ethyl acetate (9 : 1), ethyl acetate - acetone (9 : 1), and
ethyl acetate - acetone - acetic acid glacial (7 : 2 : 1). Spray reagent used was UV λ
254 nm, UV λ 366 nm, and 10% H 2SO 4 in methanol as a universal reagent.
In this monitoring extracts carried out a comparison between the chromatogram
patterns of bee pollen extract and extracts of maize pollen. From the monitoring
results obtained that there is no significant difference between the content of
compounds in bee pollen and maize pollen. In n-hexane extract contained some
brownish spots which would then be isolated and characterized, and was expected
to be a marker compound bee pollen.
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9. Characterization Of Bee Pollen From Ranca Bungur, Bogor
Figure 6 Thin layer chromatogram of n-hexane, ethyl acetate, and ethanol
extracts at stationary phase silica gel GF254, (1) n-hexane extract
with mobile phase toluene - ethyl acetate (9 : 1), (2) ethyl acetate
extract with mobile phase ethyl acetate - acetone (9 : 1), and (3)
ethanol extract with mobile phase ethyl acetate - acetone - glacial
acetic acid (7 : 2 : 1), (i) UV λ 254 nm, (ii) UV λ 366 nm, and (iii)
10% H 2SO 4 in methanol visual, (a) bee pollen, (b) maize pollen.
From the results of monitoring extract of ethanol can be seen there is a difference
between the compound extract of bee pollen and maize pollen. In the extract of bee
pollen contained blue spots under UV light at λ 366 nm Rf 0.7, while the extract of
maize pollen is not there. After addition of 10% H 2SO 4 reagent in methanol
contained brownish spotting at Rf 0 to 0.4 which allegedly is sugar derived
compounds.
In this research study focused on secondary metabolites spots with Rf 0.8 of n -
hexane extract. Furthermore, n-hexane extract was fractionated by classical
column chromatography using silica gel 60 as stationary phase and eluent n -
hexane - ethyl acetate (9 : 1). Fractions were collected by volume, 10 mL and
obtained 33 fractions. Then the monitoring fractions of bee pollen was done to the
fraction numbers 5, 10, 15, 20, 25, and 30 that conducted in TLC with the
stationary phase silica gel GF254. Mobile phase used was n-hexane - ethyl acetate
(9 : 1). From the monitoring results obtained fractions isolated compounds will be
present in early fractions, namely fraction 5. Furthermore, the monitoring was
done back in fractions of fractions 1, 2, 3, 4, 5, and 6 with the same
chromatographic system.
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10. DEVI KAMILIA A., SUKRASNO & IRDA FIDRIANNY
Figure 7 Thin layer chromatogram monitoring of the fractions with
stationary phase silica gel GF254, and mobile phase n-hexane -
ethyl acetate (9 : 1), with 10% H 2SO 4 reagent in methanol under
UV λ 366 nm, (a) bee pollen.
From the monitoring results to 1-6 fraction was obtained that the isolated
compound will be contained in fraction 3. Furthermore, the purity test was done to
determine the purity of the compound. The purity of two-dimensional TLC
performed with the stationary phase of silica gel GF254. Mobile phase used was n-
hexane - ethyl acetate (9 : 1) and toluene - ethyl acetate (7 : 3).
Figure 8 Two-dimensional thin-layer chromatogram fraction 3, with
stationary phase silica gel GF254, mobile phase n-hexane - ethyl
acetate (9 : 1) and toluene - ethyl acetate (7 : 3), with 10% H 2SO 4
reagent in methanol under light UV λ 366 nm.
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11. Characterization Of Bee Pollen From Ranca Bungur, Bogor
From the purity test results can be seen that the fractionation was done purely so
that further characterization. From UV-visible light spectrum can be seen that
there are compounds give maximum absorbance at a wavelength of 235 nm and
321 nm.
Figure 9 The spectrum of UV-visible spectrophotometry isolates in the n-hexane
solvent.
From the infrared spectrum can be seen that there are compounds present in the
hydroxy group at wave numbers 3363 cm-1 which shows the OH stretch. At wave
numbers 2923 and 2857 cm-1 show CH stretch, which means there is an aliphatic
group. At wave numbers 1735 cm-1 shows a stretching C = O, which means there
are carbonyl groups. At wave numbers 1643 cm-1 shows a stretching C = C, which
may indicate the existence of an aliphatic or aromatic group. At wave numbers
1461 and 1376 cm-1 shows the CH bending and the wave numbers of 721 cm -1
shows the bending C = C, which also can indicate the existence of aromatic group.
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12. DEVI KAMILIA A., SUKRASNO & IRDA FIDRIANNY
Figure 10 The spectrum of infrared spectrophotometry isolates in KBr pellets.
In addition, the isolate was characterized by a variety of specific reagents such as
Liebermann-Burchard for steroid/triterpenoid, anisaldehid for terpenoid, sitroborat
and AlCl 3 for flavonoid, and FeCl 3 for phenol. The isolate gave a positive reaction
with FeCl 3 reagent. From the results of this characterization can be concluded that
the isolate was a phenolic compounds.
4 Conclusion
Bee pollen was used as the test material in this research the main source of pollen
from maize plants. Bee pollen contains several nutrients i.e. carbohydrate, protein
and lipid in the number row is 56.14 ± 0.07%, 25.82 ± 0.34%, and 5.07 ±
0.12%. Total carbohydrate content in the bee pollen is higher than the levels of
carbohydrate in maize pollen. Carbohydrate component contained in bee pollen
include fructose, glucose, sucrose and other oligosaccharides, whereas in maize
pollen contained only glucose and fructose. The phytochemical screening showed
that bee pollen contains flavonoid and steroid/triterpenoid compounds. Isolates
obtained from n-hexane extract of bee pollen was thought to phenolic compounds
that contain hydroxy groups, carbonyl, aromatic and aliphatic, possibly a
conjugate of hydroxy cinnamate.
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13. Characterization Of Bee Pollen From Ranca Bungur, Bogor
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Details of author(s)
DEVI KAMILIA A
School of Pharmacy,
Institut Teknologi Bandung
West Java, Indonesia
e-mail: devi_kamilia@yahoo.com.sg
SUKRASNO
School of Pharmacy,
Institut Teknologi Bandung
West Java, Indonesia
IRDA FIDRIANNY
School of Pharmacy,
Institut Teknologi Bandung
West Java, Indonesia
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